Development Of Diabetic Nephropathy Research Articles

Circ-Luc7l Absence Attenuates Diabetic Nephropathy Progression by Reducing Mesangial Cell Excessive Proliferation, Inflammation, and Extracellular Matrix Accumulation via Mediating the miR-205-5p/Tgfbr1 Pathway.

Diabetic nephropathy (DN) threatens the survival quality of patients, with complex pathogenesis. Circular RNA (circRNA) dysregulation occurs in DN development. This work aimed to investigate the role of circ-Luc7l in DN cell models and related molecular mechanisms. The expression of circ-Luc7l, microRNA (miR)-205-5p, and transforming growth factor-beta receptor 1 (Tgfbr1) was examined by real-time quantitative PCR (RT-qPCR). Cell viability and proliferation were detected by Cell Counting Kit-8 (CCK-8) assay and EdU assay. The expression of extracellular matrix (ECM)-related markers and Tgrbr1 protein was measured by Western blot. The binding between miR-205-5p and circ-Luc7l or Tgfbr1 was validated by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, or RNA pull-down assay. Experimental animal models were established to elucidate the function of circ-Luc7l in vivo. Circ-Luc7l expression was notably enhanced in high glucose (HG)-treated mesangial cells. Knockdown of circ-Luc7l attenuated HG-induced cell proliferation, inflammation, and ECM accumulation in vitro and relieved inflammation and ECM accumulation of kidneys of diabetic mice in vivo. Circ-Luc7l targeted miR-205-5p, and miR-205-5p inhibition rescued the depletion effects of circ-Luc7l knockdown on cell proliferation, inflammation, and ECM accumulation. MiR-205-5p bound to Tgfbr1 whose expression was negatively regulated by circ-Luc7l. Tgfbr1 overexpression also rescued the depletion effects of circ-Luc7l knockdown on cell proliferation, inflammation, and ECM accumulation. In HG conditions, increased circ-Luc7l upregulated Tgfbr1 expression via targeting miR-205-5p to induce DN progression.

HINT2 may be One Clinical Significance Target for Patient with Diabetes Mellitus and Reduced ROS-Induced Oxidative Stress and Ferroptosis by MCU.

The World Health Organization (WHO) predicted that patients with diabetes around the world will increase to 600 million by 2040, of which about 1/3 will develop diabetic nephropathy (DN). Therefore, the present study aimed to uncover therapeutic effect of HINT2 and determined its possible mechanisms. Patients with diabetes mellitus and normal volunteers were enrolled at our hospital. Male C57BL/6 mice were fed with a high fat diet and injected intraperitoneally with STZ for once (100 mg/kg body weight). Mouse podocytes (MPC5) cells were induced with 20 mmol/l D-glucose. Inhibition of HINT2 mRNA expression levels in patients with DN was observed, compared with normal group. The serum of HINT2 mRNA expression was negative in correlation with blood sugar, tubulo-interstitial damage, glomerular damage score or urine protein level in patients with DN. HINT2 expression in kidney tissue of mice with DN were downregulated. HINT2 presented reduced DN and inflammation and ROS-induced oxidative stress in model of DN. HINT2 promoted ferroptosis in model of DN by mitochondrial membrane potential. HINT2 suppressed MCU expression in model of DN. HINT2 protein combined with MCU protein increased MCU protein ubiquitination. HINT2 triggers mitochondrial Ca2+ influx to increase ROS production level by MCU. Taken together, these findings demonstrated that HINT2 reduced ROS-induced Oxidative stress and ferroptosis by MCU, suggesting that HINT2 may be a feasible strategy to treat DN.

Silencing of METTL3 prevents the proliferation, migration, epithelial-mesenchymal transition, and renal fibrosis of high glucose-induced HK2 cells by mediating WISP1 in m6A-dependent manner.

Diabetic nephropathy (DN) is one of the most serious complications in diabetic patients. And m6A modifications mediated by METTL3 are involved multiple biological processes. However, the specific function and mechanism of METTL3 in DN remains unclear. DN model mice were first established with streptozotocin, and WISP1 expression was confirmed by qRT-PCR. Then the influences of WISP1 or/and METTL3 on the proliferation, migration, and epithelial-mesenchymal transition (EMT) and fibrosis-related proteins of high glucose (HG)-induced HK2 cells or HK2 cells were tested through CCK-8, wound healing, and western blot. We first revealed that WISP1 was highly expressed in renal tissues of DN model mice and HG-induced HK2 cells. Functionally, WISP1 or METTL3 silencing could weaken the proliferation, migration, EMT, and fibrosis of HG-treated HK2 cells, and WISP1 or METTL3 overexpression could induce the proliferation, migration, EMT, and fibrosis of HK2 cells. Additionally, METTL3 silencing could decrease WISP1 m6A modification, and silencing of METTL3 also could notably suppress the biological functions of HG-induced HK2 cells by downregulating WISP1. Silencing of METTL3 prevents DN development process by decreasing WISP1 with m6A modification pattern. Therefore, we suggest that METTL3/WISP1 axis might be a novel therapeutic target for DN.

Association between lipoprotein(a) plasma levels and diabetic nephropathy in Han Chinese patients with type 2 diabetes mellitus.

The goal of this study was to evaluate the relationship between serum lipoprotein(a) [Lp(a)] levels and diabetic nephropathy (DN) among Han Chinese individuals with type 2 diabetes mellitus (T2DM). This retrospective analysis comprised a consecutive case series of 767 grown-up patients with T2DM (199 among them with DN) hospitalized in the Department of Endocrinology at the The First Affiliated Hospital of Anhui Medical University from February 20220 to February 2021. Clinical data and other laboratory measurements, such as glycated hemoglobin (HbA1c), were extracted from medical records and compared among groups. Clinical characteristics according to Lp(a) quartiles were also studied. Univariate and multivariate regression analysis were used to examine the relationship between serum Lp(a) and DN. Patients with DN had a longer disease duration, higher HbA1c, higher level of Lp(a), and were more likely to have diabetic retinopathy (DR) than those without DN (P < 0.005 for each). With regard to the Lp(a) quartile group, patients with a higher Lp(a) concentration were more likely to have DN and have higher level of HbA1c during the study (P for trend < 0.005 for each). After adjusting for several confounding factors, the development of DN was significantly associated with the serum Lp(a) level (P = 0.026, comparing the 4th vs 1st quartile of Lp(a)) according to multivariate regression analysis. The receiver operating characteristic (ROC) curves for DN development using serum Lp(a) showed that the area under the receiver operating characteristic curves (AUC) was 0.590 (P < 0.001). Findings from this study demonstrated that the DN was independently associated with the serum Lp(a) level in patients with T2DM in this retrospective study.

Long non-coding RNA MEG3 silencing weakens high glucose-induced mesangial cell injury by decreasing LIN28B expression by sponging and sequestering miR-23c.

Diabetic nephropathy (DN) is a common kidney disease in diabetic patients. Long non-coding RNA maternally expressed gene 3 (MEG3) and microRNA (miR)-23c are reported to be implicated in DN development. Nevertheless, it is unclear that the molecular mechanism between MEG3 and miR-23c in DN remains unclear. Human mesangial cells (HMCs) were treated with high glucose (HG) to simulate the DN status in vitro. Expression of MEG3 and miR-23c was measured. Effects of MEG3 silencing on HG-stimulated HMC injury were determined. The relationship between MEG3 and miR-23c was verified by the dual-luciferase reporter and RNA immunoprecipitation assays. MEG3 was overexpressed in serums from DN patients and HG-stimulated HMCs. MEG3 knockdown weakened HG-stimulated HMC proliferation, extracellular matrix (ECM) accumulation, and inflammation. MEG3 regulated lin-28 homolog B (LIN28B) expression through adsorbing miR-23c. MiR-23c inhibitor reversed MEG3 knockdown-mediated effects on HG-stimulated HMC proliferation, ECM accumulation, and inflammation. LIN28B overexpression overturned miR-23c mimic-mediated effects on HG-stimulated HMC proliferation, ECM accumulation, and inflammation. MEG3 regulated HMC injury via regulation of the miR-23c/LIN28B axis in DN, which can help us better understand the mechanism of DN mediated by MEG3.

Upregulation of UHRF1 Promotes PINK1-mediated Mitophagy to Alleviates Ferroptosis in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a common diabetic complication. Studies show that mitophagy inhibition induced-ferroptosis plays a crucial role in DN progression. UHRF1 is associated with mitophagy and is highly expression in DN patients, however, the effect of UHRF1 on DN is still unclear. Thus, in this study, we aimed to investigate whether UHRF1 involves DN development by the mitophagy/ferroptosis pathway. We overexpressed UHRF1 using an adeno-associated virus 9 (AAV9) system in high-fat diet/streptozotocin-induced diabetic mice. Renal function index, pathological changes, mitophagy factors, and ferroptosis factors were detected in vivo. High-glucose cultured human renal proximal tubular (HK-2) cells were used as in vitro models to investigate the mechanism of UHRF1 in DN. We found that diabetic mice exhibited kidney damage, which was alleviated by UHRF1 overexpression. UHRF1 overexpression promoted PINK1-mediated mitophagy and inhibited the expression of thioredoxin interacting protein (TXNIP), a factor associated with mitochondrial dysfunction. Additionally, UHRF1 overexpression alleviated lipid peroxidation and free iron accumulation, and upregulated the expression of GPX4 and Slc7a11, indicating the inhibition effect of UHRF1 overexpression on ferroptosis. We further investigated the mechanism of UHRF1 in the mitophagy/ferroptosis pathway in DN. We found that UHRF1 overexpression promoted PINK1-mediated mitophagy via inhibiting TXNIP expression, thus suppressing ferroptosis. These findings confirmed that upregulation of UHRF1 expression alleviates DN, indicating that UHRF1 has a reno-protective effect against DN.

Identification and validation of voltage-dependent anion channel 1-related genes and immune cell infiltration in diabetic nephropathy.

This study investigated the roles of voltage-dependent anion channel 1-related differentially expressed genes (VRDEGs) in diabetic nephropathy (DN). We downloaded two datasets from patients with DN, namely, GSE30122 and GSE30529, from the Gene Expression Omnibus database. VRDEGs associated with DN were obtained from the intersection of voltage-dependent anion channel 1-related genes from the GeneCards database, and differentially expressed genes were screened according to group (DN/healthy) in the two datasets. The enriched pathways of the VRDEGs were analyzed. Hub genes were selected using a protein-protein interaction network, and their predictive value was verified through receiver operating characteristic curve analysis. The CIBERSORTx software examined hub genes and immune cell infiltration associations. The protein expression of hub genes was verified through immunohistochemistry in 16-week-old db/db mice for experimentation as a model of type 2 DN. Finally, potential drugs targeting hub genes that inhibit DN development were identified. A total of 57 VRDEGs were identified. The two datasets showed high expression of the PI3K, Notch, transforming growth factor-β, interleukin-10 and interleukin-17 pathways in DN. Five hub genes (ITGAM, B2M, LYZ, C3 and CASP1) associated with DN were identified and verified. Immunohistochemistry showed that the five hub genes were highly expressed in db/db mice, compared with db/m mice. The infiltration of immune cells was significantly correlated with the five hub genes. Five hub genes were significantly correlated with immune cell infiltration and might be crucial to DN development. This study provides insight into the mechanisms involved in the pathogenesis of DN.

Serum Pentraxin-3 as a Potential Biomarker for Early Detection of Diabetic Nephropathy in Type 2 Diabetes Mellitus: A Cross-Sectional Study

This cross-sectional study aimed to assess the utility of serum Pentraxin-3 (PTX-3) as a biomarker for the early identification of diabetic nephropathy (DN) in patients with Type 2 Diabetes Mellitus (T2DM). A total of 90 participants, including 30 with T2DM and nephropathy, 30 with T2DM but without nephropathy, and 30 controls, were enrolled. Serum PTX-3 levels were measured, and correlations with clinical parameters were analyzed. The results revealed a significant elevation in serum PTX-3 levels in DN patients with T2DM. PTX-3 exhibited positive correlations with age, disease duration, blood urea, and serum creatinine, indicating its potential relevance to DN development and renal function. Conversely, non-significant negative correlations were observed with BMI and estimated glomerular filtration rate (eGFR). These findings suggest that serum PTX-3 could serve as a valuable biomarker for the early detection of diabetic nephropathy in Type 2 Diabetes Mellitus, potentially aiding in timely intervention and improved patient care.&#x0D; Highlights : &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; Serum PTX-3 levels significantly elevated in Type 2 Diabetes patients with nephropathy, highlighting its potential as an early diagnostic biomarker.&#x0D; Positive correlations observed between PTX-3, age, disease duration, blood urea, and serum creatinine, suggesting its relevance to DN development and renal function.&#x0D; Serum PTX-3 holds promise for timely identification of diabetic nephropathy, facilitating improved patient care.&#x0D; &#x0D; Keywords : Diabetic Nephropathy, Pentraxin-3, Type 2 Diabetes, Biomarker. Early Detection&#x0D; &#x0D; &#x0D; &#x0D;  &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D;  &#x0D;

Persistence and expansion of hypoxia detected by pimonidazole adduct immunostaining during progression of diabetic nephropathy in diabetic mice.

Hypoxia and oxidative stress are considered to be underlying factors in the deterioration of renal function and pathogenesis in acute kidney injury (AKI) and chronic kidney disease, including diabetic nephropathy (DN). However, the long-term role of hypoxia in DN is unknown. Here, we investigated the distribution, severity, and time course of hypoxia during DN development in our well-established severely diabetic transgenic (Tg) DN mouse model that mimics human DN up to 80 wk of age, using pimonidazole adduct immunohistochemistry. The relationship between pimonidazole adduct distribution and hypoxia-inducible factor (HIF) expression was also examined. We found 1) persistent pimonidazole immunostaining mainly in the outer zone of the outer medulla, extending into the inner zone, 2) significant expansion of area and intensity up to 40 wk of age, and 3) characteristic subcellular localization mainly at apical sites in vesicular form by laser scanning microscopy of thin slices. The distribution of pimonidazole adducts was different from that of HIF reported previously, indicating that hypoxia does not directly contribute to persistent abnormal HIF expression. These results suggest that pimonidazole adducts produced under low [Formula: see text] conditions are sustained by a mechanism distinct from direct ischemia. We propose that in the long course of DN development, persistent hyperfiltration and hyperexcretion of glucose, albumin, and water increase metabolism and energy expenditure in the tubules, and such chronic stimulation leads to relative ischemia and local hypoxia, which may contribute in part to the loss of nephrons.NEW & NOTEWORTHY This study provides new insights into hypoxia during the long course of diabetic nephropathy development. Hypoxia was persistently localized only in limited areas and its distribution differed significantly from that of hypoxia-inducible factors. These findings suggests that in the long course of diabetic nephropathy development, increased energy requirements and limited blood supply may lead to relative ischemia and induction of local and persistent hypoxia, which may contribute in part to the loss of nephrons.

Genotypic Analysis of COL4A1 Gene in Diabetic Nephropathy and Type 2 Diabetes Mellitus Patients: A Comparative Genetic Study.

Diabetic nephropathy (DN) is specified by microalbuminuria, glomerular lesions, and renal fibrosis leading to end-stage renal disease. The pathophysiology of DN is multifactorial as a result of gene-environment interaction. Clinical studies suggested that gene mutations affect various pathways involved in DN, including extracellular matrix (ECM). During chronic hyperglycemia, collagen type-4-mediated ECM overproduction occurs, leading to renal fibrosis and DN development. In this study, COL4A1 gene variant rs605143 (G/A) was analyzed in diabetes and DN patients from the study population. We genotyped 386 study subjects, comprising 120 type 2 diabetes mellitus (T2DM) patients, 120 DN, and 146 healthy controls. All study subjects were analyzed for biochemical assays by commercially available kits and genotypic analysis by polymerase chain reaction-restriction fragment length polymorphism and confirmed by Sanger sequencing. Statistical analyses were done using SPSS and GraphPad. Anthroclinicopathological parameters showed a significant association between T2DM and DN. Genotype AA of COL4A1 gene variant rs605143 (G/A) showed a significant association with T2DM and DN compared with controls with 5.87- and 8.01-folds risk, respectively. Mutant allele A also significantly associated with T2DM and DN independently compared with healthy controls with 2.29- and 2.81-time risk in the study population. This study's findings suggested that COL4A1 gene variant rs605143 (G/A) can be used as predictive biomarkers for T2DM and DN independently. However, this gene variant needs to be analyzed in a large sample to explore the shared genetic association between T2DM and DN.

Identification of ferroptosis-related genes as potential diagnostic biomarkers for diabetic nephropathy based on bioinformatics.

Objective: This study investigated to probe ferroptosis-related diagnostic biomarkers and underlying molecular mechanisms in Diabetic nephropathy (DN). Methods: GSE30122 and GSE1009 from GEO database were used as training and verification sets, respectively, to screen differentially expressed ferroptosis-related genes (FRGs). These genes were further analyzed using GO, KEGG, and GSEA methods, and screened with PPI, LASSO, and SVM-RFE to identify ferroptosis-related diagnostic biomarkers for DN. A diagnostic model was established using the Glm function and verified with ROC curve. The relationship between these biomarkers and immune cell was analyzed, and qRT-PCR and Western blot were used to detect the expression of these biomarkers in kidney tissues and identify the effect of TP53 on DN development. Results: Fifty one differentially expressed FRGs were enriched in bioprocesses such as p53 signaling pathway, oxidative stress and chemical stress response, and mTOR signaling pathway. TP53, RB1, NF2, RRM2, PRDX1, and CDC25A were identified as ferroptosis-related diagnostic biomarkers for DN. TP53 showed the most differential expression. ROC analysis showed that AUC values of TP53, RB1, NF2, RRM2, PRDX1, and CDC25A were 0.751, 0.705, 0.725, 0.882, 0.691, and 0.675, respectively. The AUC value of DN diagnosis model was 0.939 in training set and 1.000 in verification set. qRT-PCR results confirmed significant differences in these six biomarkers between DN and normal kidney tissue (p < 0.05), and correlation analysis showed that five biomarkers were significantly correlated with infiltrating immune cells (p < 0.05). Furthermore, western blots showed that TP53 promotes apoptosis through PI3K-AKT signaling in DN. Conclusion: TP53, RB1, NF2, RRM2, PRDX1, and CDC25A have potential as diagnostic biomarkers for DN. The diagnostic model containing the above six biomarkers performs well in the diagnosis of DN. Five of the six biomarkers are strongly associated with several infiltrating immune cells. TP53 may play an essential role in the development of DN.

17-LB: Diabetic Nephropathy Prediction with Machine-Learning Models for Patients with Type 1 Diabetes

Cardiovascular disease (CVD) is the leading cause of global mortality in type 1 Diabetes (T1D). This risk remains largely under-managed, leading to a future significant CVD burden. Diabetic nephropathy (DN) is considered a risk factor for CVD and overall mortality in patients with T1D. Identifying risk factors that contribute to the development and progression of DN is therefore important in T1D. Early identification of DN may support earlier intervention for patients with T1D and in turn, reduce future risk for CVD. Using the T1D Exchange Registry database of individuals with T1D in the USA, we sought to develop a machine-learning algorithm to predict patients with DN. With performance metrics of F1-score = 0.67, AUC = 0.78, in our Random Forest model, and F1-score= 0.66, AUC = 0.77 in our Logistics Regression model demonstrates the potential to assist in mitigating DN development and progression. Disclosure S. Sripada: None. S. Sripada: None. S. Belapurkar: Research Support; Dexcom, Inc., Tandem Diabetes Care, Inc. Other Relationship; T1D Exchange. Research Support; Medtronic.

Roles of microRNA-192 in diabetic nephropathy: the clinical applications and mechanisms of action.

Diabetic nephropathy (DN) is one of the most common and intractable microvascular complications of diabetes worldwide, serving as the main cause of terminal renal disease. Due to the lack of early specific symptoms and diagnostic markers, DN severely threatens the sufferer's life. MicroRNA-192 (miR-192) was early identified in human renal cortical tissue and stored and excreted in urine as microvesicles. MiR-192 was found to be involved in the development of DN. For the first time, the present review summarized all the current evidence on the topic of the roles of miR-192 in DN. Finally, 28 studies (ten clinical trials and eighteen experimental studies) were eligible for thorough reviewing. Most of the clinical trials (7/10, 70%) indicated miR-192 might be a protective factor for DN development and progression, while the majority of experimental studies (14/18, 78%) suggested miR-192 might be a pathogenic factor for DN. Mechanistically, miR-192 interacts with various direct targeted proteins (i.e., ZEB1, ZEB2, SIP1, GLP1R, and Egr1) and signaling cascades (i.e., SMAD/TGF-β and PTEN/PI3K/AKT), together contribute to the pathogenesis of DN through epithelial-to-mesenchymal transition (EMT), extracellular matrix deposition, and fibrosis formation. The current review highlights the dual role of miR-192 in the development of DN. Low serum miR-192 expression could be applied for the early prediction of DN (the early stage of DN), while the high miR-192 level in renal tissues and urine may imply the progression of DN (the late stage of DN). Further investigations are still warranted to illustrate this inconsistent phenomenon, which may facilitate promoting the therapeutic applications of miR-192 in predicting and treating DN.

Profilin1 Promotes Renal Tubular Epithelial Cell Apoptosis in Diabetic Nephropathy Through the Hedgehog Signaling Pathway.

Profilin-1 (PFN1) regulates the dynamic balance of actin and plays an important role in cell functions as a hub protein in signaling molecule interaction networks. Dysregulation of PFN1 is related to pathologic kidney diseases. Diabetic nephropathy (DN) was recently reported as an inflammatory disorder, however, the molecular mechanisms of PFN1 in DN remain unclear. Therefore, the present study was conducted to explore the molecular and bioinformatic characteristics of PFN1 in DN. Bioinformatics analyses were performed on the chip of database in DN kidney tissues. A cellular model of DN was established in human renal tubular epithelial cells (HK-2) induced by high glucose. The PFN1 gene was overexpressed or knocked-down to investigate its function in DN. Flow cytometry was used to detect cell proliferation and apoptosis. PFN1 and proteins in the related signaling pathways were evaluated by Western blotting. The expression of PFN1 was significantly increased in DN kidney tissues (P < 0.001) and was correlated with a high apoptosis-associated score (Pearson's correlation = 0.664) and cellular senescence-associated score (Pearson's correlation = 0.703). PFN1 protein was mainly located in cytoplasm. Overexpression of PFN1 promoted apoptosis and blocked the proliferation of HK-2 cells treated with high levels of glucose. Knockdown of PFN1 led to the opposite effects. Additionally, we found that PFN1 was correlated with the inactivation of the Hedgehog signaling pathway in HK-2 cells treated with high levels of glucose. PFN1 might play an integral role in the regulation of cell proliferation and apoptosis during DN development by activating the Hedgehog signaling pathway. This study provided molecular and bioinformatic characterizations of PFN1, and contributed to the understanding of the molecular mechanisms leading to DN.

Identifying C1QB, ITGAM, and ITGB2 as potential diagnostic candidate genes for diabetic nephropathy using bioinformatics analysis.

Diabetic nephropathy (DN), the most intractable complication in diabetes patients, can lead to proteinuria and progressive reduction of glomerular filtration rate (GFR), which seriously affects the quality of life of patients and is associated with high mortality. However, the lack of accurate key candidate genes makes diagnosis of DN very difficult. This study aimed to identify new potential candidate genes for DN using bioinformatics, and elucidated the mechanism of DN at the cellular transcriptional level. The microarray dataset GSE30529 was downloaded from the Gene Expression Omnibus Database (GEO), and the differentially expressed genes (DEGs) were screened by R software. We used Gene Ontology (GO), gene set enrichment analysis (GSEA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to identify the signal pathways and genes. Protein-protein interaction (PPI) networks were constructed using the STRING database. The GSE30122 dataset was selected as the validation set. Receiver operating characteristic (ROC) curves were applied to evaluate the predictive value of genes. An area under curve (AUC) greater than 0.85 was considered to be of high diagnostic value. Several online databases were used to predict miRNAs and transcription factors (TFs) capable of binding hub genes. Cytoscape was used for constructing a miRNA-mRNA-TF network. The online database 'nephroseq' predicted the correlation between genes and kidney function. The serum level of creatinine, BUN, and albumin, and the urinary protein/creatinine ratio of the DN rat model were detected. The expression of hub genes was further verified through qPCR. Data were analyzed statistically using Student's t-test by the 'ggpubr' package. A total of 463 DEGs were identified from GSE30529. According to enrichment analysis, DEGs were mainly enriched in the immune response, coagulation cascades, and cytokine signaling pathways. Twenty hub genes with the highest connectivity and several gene cluster modules were ensured using Cytoscape. Five high diagnostic hub genes were selected and verified by GSE30122. The MiRNA-mRNA-TF network suggested a potential RNA regulatory relationship. Hub gene expression was positively correlated with kidney injury. The level of serum creatinine and BUN in the DN group was higher than in the control group (unpaired t test, t=3.391, df=4, p=0.0275, r=0.861). Meanwhile, the DN group had a higher urinary protein/creatinine ratio (unpaired t test, t=17.23, df=16, p<0.001, r=0.974). QPCR results showed that the potential candidate genes for DN diagnosis included C1QB, ITGAM, and ITGB2. We identified C1QB, ITGAM and ITGB2 as potential candidate genes for DN diagnosis and therapy and provided insight into the mechanisms of DN development at transcriptome level. We further completed the construction of miRNA-mRNA-TF network to propose potential RNA regulatory pathways adjusting disease progression in DN.

Endoplasmic reticulum stress-triggered ferroptosis via the XBP1-Hrd1-Nrf2 pathway induces EMT progression in diabetic nephropathy.

Diabetic nephropathy (DN) is characterized by tubulointerstitial fibrosis caused by epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. Although ferroptosis promotes DN development, the specific pathological process that is affected by ferroptosis in DN remains unclear. Herein, EMT-related changes, including increased α-smooth muscle actin (α-SMA) and Vimentin expression and decreased E-cadherin expression, were observed in the renal tissues of streptozotocin-induced DN mice and high glucose-cultured human renal proximal tubular (HK-2) cells. Treatment with ferrostatin-1 (Fer-1) ameliorated these changes and rescued renal pathological injury in diabetic mice. Interestingly, endoplasmic reticulum stress (ERS) was activated during EMT progression in DN. Inhibiting ERS improved the expression of EMT-associated indicators and further rescued the characteristic changes in ferroptosis caused by high glucose, including reactive oxygen species (ROS) accumulation, iron overload, increased lipid peroxidation product generation, and reduced mitochondrial cristae. Moreover, overexpression of XBP1 increased Hrd1 expression and inhibited NFE2-related factor 2 (Nrf2) expression, which could enhance cell susceptibility to ferroptosis. Co-immunoprecipitation (Co-IP) and ubiquitylation assays indicated that Hrd1 interacted with and ubiquitinated Nrf2 under high-glucose conditions. Collectively, our results demonstrated that ERS triggers ferroptosis-related EMT progression through the XBP1-Hrd1-Nrf2 pathway, which provides new insights into potential mechanisms for delaying EMT progression in DN.

Nephroprotective effect of milkfish, patin, and snakehead fish oil by suppressing inflammation and oxidative stress in diabetic rats

Diabetic nephropathy (DN) has been linked to a number of long-term problems caused by diabetes mellitus. Inflammatory and oxidative stress pathways contribute to DN development and progression. Many studies have shown the preventive advantages of diets rich in substances like anti-inflammatory and antioxidant elements like omega-3 fatty acids (n-3 FA) in preventing DN. Milkfish (Chanos chanos F.), patin (Pangasius micronema Blkr.), and snakehead fish (Chana striata Bloch) are types of fish oils that are known to contain n-3 FA. This study aims to prove the nephroprotective effect of the three types of fish oil in a rat model of diabetes mellitus. Thirty male rats were used in this study. The animals were randomly divided into six groups (n = 5): the non-diabetic group, the diabetes mellitus group, the diabetic with 150 mg/kg metformin orally group, the diabetic with 1000 mg/kg milkfish oil orally group, the diabetic with 1000 mg/kg patin fish oil orally group, and the diabetic with 1000 mg/kg snakehead fish oil orally group. Diabetes models were induced using 65 mg/kg streptozotocin and 230 mg/kg nicotinamide intraperitoneally. The test was carried out for 8 weeks, followed by the observation of the biochemical profiles of blood, urine, oxidative stress, and the immunohistochemistry of the kidneys. A normally and homogeneously distributed test followed by a one-way analysis of variance (ANOVA) and the LSD post hoc test were used to look at the data. At p≤0.05, the data was considered statistically significant. The results showed that serum creatinine levels did not differ significantly after the administration of milkfish, catfish, and snakehead fish oil for 8 weeks (p≥0.05). Different results were shown where the levels of serum BUN, uric acid, urine urea, and microalbumin urine were significantly different after administration of the three types of fish oil (p≤0.05). The same results were shown in oxidative stress (superoxide dismutase, glutathione, and malondialdehyde) and inflammation (interleukin-6 and tumor necrosis factor-α) profiles (p≤0.05). The conclusion is that milkfish, patin, and snakehead fish oils have moderate nephroprotection by suppressing inflammation and oxidative stress.

Integrative analyses of biomarkers and pathways for diabetic nephropathy.

Background: Diabetic nephropathy (DN) is a widespread diabetic complication and a major cause of terminal kidney disease. There is no doubt that DN is a chronic disease that imposes substantial health and economic burdens on the world's populations. By now, several important and exciting advances have been made in research on etiopathogenesis. Therefore, the genetic mechanisms underlying these effects remain unknown. Methods: The GSE30122, GSE30528, and GSE30529 microarray datasets were downloaded from the Gene Expression Omnibus database (GEO). Analyses of differentially expressed genes (DEGs), enrichment of gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were performed. Protein-protein interaction (PPI) network construction was completed by the STRING database. Hub genes were identified by Cytoscape software, and common hub genes were identified by taking intersection sets. The diagnostic value of common hub genes was then predicted in the GSE30529 and GSE30528 datasets. Further analysis was carried out on the modules to identify transcription factors and miRNA networks. As well, a comparative toxicogenomics database was used to assess interactions between potential key genes and diseases associated upstream of DN. Results: Samples from 19 DNs and 50 normal controls were identified in the GSE30122 dataset. 86 upregulated genes and 34 downregulated genes (a total of 120 DEGs). GO analysis showed significant enrichment in humoral immune response, protein activation cascade, complement activation, extracellular matrix, glycosaminoglycan binding, and antigen binding. KEGG analysis showed significant enrichment in complement and coagulation cascades, phagosomes, the Rap1 signaling pathway, the PI3K-Akt signaling pathway, and infection. GSEA was mainly enriched in the TYROBP causal network, the inflammatory response pathway, chemokine receptor binding, the interferon signaling pathway, ECM receptor interaction, and the integrin 1 pathway. Meanwhile, mRNA-miRNA and mRNA-TF networks were constructed for common hub genes. Nine pivotal genes were identified by taking the intersection. After validating the expression differences and diagnostic values of the GSE30528 and GSE30529 datasets, eight pivotal genes (TYROBP, ITGB2, CD53, IL10RA, LAPTM5, CD48, C1QA, and IRF8) were finally identified as having diagnostic values. Conclusion: Pathway enrichment analysis scores provide insight into the genetic phenotype and may propose molecular mechanisms of DN. The target genes TYROBP, ITGB2, CD53, IL10RA, LAPTM5, CD48, C1QA, and IRF8 are promising new targets for DN. SPI1, HIF1A, STAT1, KLF5, RUNX1, MBD1, SP1, and WT1 may be involved in the regulatory mechanisms of DN development. Our study may provide a potential biomarker or therapeutic locus for the study of DN.

Clinico-pathological features of diabetic and non-diabetic renal diseases in type 2 diabetic patients: a retrospective study from a 10-year experience in a single center.

To compare clinical and pathological characteristics as well as prognosis between diabetic nephropathy (DN) and non-diabetic renal disease (NDRD) so as to explore potential diagnostic criteria of DN and provide some guidance for the treatment of type 2 diabetes mellitus (T2DM) patients with kidney involvement. T2DM patients with renal impairment who underwent kidney biopsy were included in this study, who were classified into 3 groups (DN, NDRD, DN with NDRD) based on their renal pathological diagnosis. Baseline clinical characteristics as well as follow-up data were collected and analyzed among 3 groups. Logistic regression was performedto determine the best predictors for DN diagnosis. Additional 34 MN patients without diabetes were enrolled by propensity score matching method to compare serum PLA2R antibody titer and kidney outcomes between diabetic MN patients and MN alone. Among 365 patients with type 2 diabetes who underwent kidney biopsy, 179 (49.0%) patients were diagnosed with NDRD alone and 37 (10.1%) patients with NDRD combined DN. Risk factors for DN development in T2DM patients were longer time since diabetes diagnosis, higher level of serum creatinine, absence of hematuria and presence of diabetic retinopathy by multivariate analysis. Lower rate of proteinuria remission and higher risk of renal progression were observed in DN group compared with NDRD group. Membranous nephropathy was the most common NDRD in diabetic patients. There was no difference in serum PLA2R antibody positiveness or titer between MN patients with or without T2DM. There was lower remission rate but similar renal progression in diabetic MN when age, gender, baseline eGFR, albuminuria and IFTA score were adjusted. Non-diabetic renal disease is not uncommon in T2DM patients with renal impairment, which has better prognosis with proper treatment. Coexisting diabetic status does not exert negative impact on renal progression in MN patients, and immunosuppressive agents should be administered when necessary.

Long non-coding RNA DLX6-AS1 is the key mediator of glomerular podocyte injury and albuminuria in diabetic nephropathy by targeting the miR-346/GSK-3β signaling pathway

Progressive albuminuria is the primary clinical symptom of diabetic nephropathy (DN), leading to a gradual decline in kidney function. DLX6-AS1 was the first reported long non-coding RNA (lncRNA) to participate in organogenesis and play crucial roles in the brain or neural cell development. Herein, we investigated the DLX6-AS1 (Dlx6-os1 in mice) role in DN pathogenesis. We found that DLX6-AS1 expression in DN patients correlated with the extent of albuminuria. Dlx6-os1 overexpression induced cellular damage and inflammatory responses in cultured podocytes through miR-346-mediated regulation of the GSK-3β pathway. In various established diabetic and newly developed knockout mouse models, Dlx6-os1 knockdown/knockout significantly reduced podocyte injury and albuminuria. The Dlx6-os1 effects were remarkably modulated by miR-346 mimics or mutants and significantly diminished in podocyte-specific GSK-3β-knockout mice. Thus, DLX6-AS1 (Dlx6-os1) promotes DN development by accelerating podocyte injury and inflammation through the upregulation of the GSK-3β pathway, providing a novel molecular target for DN therapy.